Violaine K. Harris

Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is associated with irreversible disability in a significant proportion of patients. At present, there is no treatment to halt or reverse the progression of established disability. In an effort to develop cell therapy-based strategies for progressive MS, we investigated the pre-clinical efficacy of bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) as an autologous source of stem cells. MSC-NPs consist of a subpopulation of bone marrow MSCs with neural progenitor and immunoregulatory properties, and a reduced capacity for mesodermal differentiation, suggesting that this cell population may be appropriate for clinical application in the CNS. We investigated whether MSC-NPs could promote repair and recovery after intrathecal injection into mice with EAE. Multiple injections of MSC-NPs starting at the onset of the chronic phase of disease improved neurological function compared to controls, whereas a single injection had no effect on disease scores. Intrathecal injection of MSC-NPs correlated with reduced immune cell infiltration, reduced area of demyelination, and increased number of endogenous nestin-positive progenitor cells in EAE mice. These observations suggest that MSC-NPs may influence the rate of repair through effects on endogenous progenitors in the spinal cord. This study supports the use of autologous MSC-NPs in MS patients as a means of promoting CNS repair.

Disease Biomarkers in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disorder of the brain and spinal cord that predominantly affects white matter. MS has a variable clinical presentation and has no ‘diagnostic’ laboratory test; this often results in delays to definite diagnosis. In confronting the disease, early diagnosis and appropriate, timely therapeutic intervention are critical factors in ensuring favorable long-term outcomes.The availability of reliable biomarkers could radically alter our management of MS at critical phases of the disease spectrum. Identification of markers that could predict the development of MS in high-risk populations would allow for intervention strategies that may prevent evolution to definite disease. Work with anti-myelin antibodies and the ongoing analysis of microarray gene expression have thus far not yielded biomarkers that predict future disease development. Similarly, extensive studies with serum and cerebrospinal fluid (CSF) have not yielded a disease-specific and sensitive diagnostic biomarker for MS. Establishment of disease diagnosis always leads to questions about long-term prognosis because in an individual patient the natural history of the disease is clinically unpredictable. Biomarkers that correlate with myelin loss, spinal cord disease, grey matter and subcortical demyelination need to be developed in order to accurately predict the disease course. The bulk of effort in biomarker development in MS has been concentrated in the area of monitoring disease activity. At present, a disease ‘activation’ panel of CSF biomarkers would include the following: interleukin-6 or its soluble receptor, nitric oxide and nitric oxide synthase, osteopontin, and fetuin-A. Although disease activity in MS is predominantly inflammatory, disease progression is likely to be the result of neurodegeneration. Therefore, the roles of proteins indicative of neuronal, axonal, and glial loss such as neurofilaments, tau, 14-3-3 proteins, and N-acetylaspartate are all under investigation, as are proteins affecting remyelination and regeneration, such as Nogo-A. With the increasing awareness of cognition dysfunction in MS, molecules such as apolipoprotein and proteins in the amyloid precursor protein pathway implicated in dementia are also being examined.Serum biomarkers that help monitor therapeutic efficacy such as the titer of antibody to β-interferon, a first-line medication in MS, are established in clinical practice. Ongoing work with biomarkers that reflect drug bioavailability and factors that distinguish between medication responders and nonresponders are also under investigation.The discovery of new biomarkers relies on applying advances in proteomics along with microarray gene and antigen analysis and will hopefully result in the establishment of specific biomarkers for MS.

Mesenchymal Stem Cells Enhance the Engraftment and Myelinating Ability of Allogeneic Oligodendrocyte Progenitors in Dysmyelinated Mice

Multiple sclerosis is an autoimmune disease characterized by demyelination and axonal loss throughout the central nervous system. No regenerative treatment exists for patients who fail to respond to conventional immunosuppressive and immunomodulating drugs. In this scenario, stem cell therapy poses as a rational approach for neurological regeneration. Transplantation of embryonic-derived oligodendrocyte progenitor cells (OPCs) has been shown to promote remyelination and ameliorate animal models of neurodegenerative diseases. However, its therapeutic application is limited due to potential transplant rejection. In multiple sclerosis, an added concern is that transplant rejection would be most pronounced at sites of previous lesions, exacerbating a hyperactive immune response which could prevent remyelination and precipitate additional demyelination. Routine systemic immunosuppression may not be sufficient to prevent transplant rejection-associated immune reactions in the cerebral microenvironment. Mesenchymal stem cells (MSCs), due to their homing properties and inherent immunosuppressive nature, are a promising tool for clinical application targeted toward immunosuppression at sites of injury. In this study, we used a co-transplantation strategy to investigate the effect of syngeneic MSCs on the survival and remyelination abilities of allogeneic OPCs in adult nonimmunosuppressed shiverer mice. At all time points examined, cotransplantation with MSCs increased OPC engraftment, migration, and maturation in myelinating oligodendrocytes, which produced widespread myelination in the host corpus callosum. In addition, MSCs reduced microglia activation and astrocytosis in the brain of transplanted animals as well as T-cell proliferation in vitro. These data suggest that combining the immunomodulatory and trophic properties of MSCs with the myelinating ability of OPCs might be a suitable strategy for promoting neurological regeneration in demyelinating diseases.

Characterization of Autologous Mesenchymal Stem Cell-Derived Neural Progenitors as a Feasible Source of Stem Cells for Central Nervous System Applications in Multiple Sclerosis

Bone marrow mesenchymal stem cell‐derived neural progenitors (MSC‐NPs) are a potential therapeutic source of cells that have been shown to be efficacious in a preclinical model of multiple sclerosis (MS). To examine the feasibility of using MSC‐NPs as an autologous source of cells to promote central nervous system (CNS) repair in MS, this study characterized human MSC‐NPs from a panel of both MS and non‐MS donors. Expanded MSCs showed similar characteristics in terms of growth and cell surface phenotype, regardless of the donor disease status. MSC‐NPs derived from all MSCs showed a consistent pattern of gene expression changes that correlated with neural commitment and increased homogeneity. Furthermore, the reduced expression of mesodermal markers and reduced capacity for adipogenic or osteogenic differentiation in MSC‐NPs compared with MSCs suggested that MSC‐NPs have reduced potential of unwanted mesodermal differentiation upon CNS transplantation. The immunoregulatory function of MSC‐NPs was similar to that of MSCs in their ability to suppress T‐cell proliferation and to promote expansion of FoxP3‐positive T regulatory cells in vitro. In addition, MSC‐NPs promoted oligodendroglial differentiation from brain‐derived neural stem cells that correlated with the secretion of bioactive factors. Our results provide a set of identity characteristics for autologous MSC‐NPs and suggest that the in vitro immunoregulatory and trophic properties of these cells may have therapeutic value in the treatment of MS.

Cerebrospinal fluid fetuin-A is a biomarker of active multiple sclerosis

Background: There is an urgent need for biomarkers in multiple sclerosis (MS) that can reliably measure ongoing disease activity relative to inflammation, neurodegeneration, and demyelination/remyelination. Fetuin-A was recently identified as a potential biomarker in MS cerebrospinal fluid (CSF). Fetuin-A has diverse functions, including a role in immune pathways. Objective: The objective of this research is to investigate whether fetuin-A is a direct indicator of disease activity. Methods: We measured fetuin-A in CSF and plasma of patients with MS and correlated these findings to clinical disease activity and natalizumab response. Fetuin-A expression was characterized in MS brain tissue and in experimental autoimmune encephalomyelitis (EAE) mice. We also examined the pathogenic role of fetuin-A in EAE using fetuin-A-deficient mice. Results: Elevated CSF fetuin-A correlated with disease activity in MS. In natalizumab-treated patients, CSF fetuin-A levels were reduced one year post-treatment, correlating with therapeutic response. Fetuin-A was markedly elevated in demyelinated lesions and in gray matter within MS brain tissue. Similarly, fetuin-A was elevated in degenerating neurons around demyelinated lesions in EAE. Fetuin-A-deficient mice demonstrated delayed onset and reduced severity of EAE symptoms. Conclusions: Our results show that CSF fetuin-A is a biomarker of disease activity and natalizumab response in MS. Neuronal expression of fetuin-A suggests that fetuin-A may play a pathological role in the disease process.

Bri2-23 is a potential cerebrospinal fluid biomarker in multiple sclerosis

To identify potential multiple sclerosis (MS)-specific biomarkers, we used a proteomic approach to screen cerebrospinal fluid (CSF) from 40 MS patients and 13 controls. We identified seven proteins (Beta-2-microglobulin, Bri2-23, Fetuin-A, Kallikrein-6, Plasminogen, Ribonuclease-1, and Transferrin) that had significantly altered levels in MS compared to controls. Clinical subgroup analysis revealed that decreased CSF levels of Bri2-23, a peptide cleaved from Bri2, were significantly associated with patients having cerebellar dysfunction and cognition impairment. Furthermore, expression levels of Bri2 were specifically decreased in the cerebellum compared to other areas of same brain in MS but not in controls, suggesting that decreased cerebellar Bri2 expression may play a role in cerebellar dysfunction. The association with cognition impairment is also of interest because Bri2 is linked to the amyloid processing pathway in the brain. CSF levels of Bri2-23 may serve as a biomarker of these functions in MS and merits further investigation.

Clinical safety of intrathecal administration of mesenchymal stromal cell-derived neural progenitors in multiple sclerosis.

BACKGROUND AIMS There is a critical unmet need to develop regenerative therapies for the demyelinating disease multiple sclerosis (MS). We previously characterized the immunoregulatory and trophic properties of neural progenitors derived from bone marrow mesenchymal stromal cells (MSC-NPs) and established that cells derived from MS and non-MS patients alike were therapeutically viable. In an experimental model of MS, intrathecal MSC-NP injection resulted in disease amelioration with decreased T-cell infiltration, and less severe lesion pathology associated with recruitment of resident progenitors to inflammatory sites. In this pilot feasibility study, we investigated safety and dosing of intrathecal MSC-NP therapy in six patients with MS. METHODS Patients with progressive MS and advanced disability who were refractory to all other conventional MS treatments were enrolled in the study. For each dose, MSC-NP cells were cultured from autologous MSCs and tested for quality control before intrathecal administration. Patients were evaluated for adverse events and neurological status to assess safety of the treatment. RESULTS Six patients with progressive MS were treated with between 2 and 5 intrathecal injections of escalating doses of autologous MSC-NPs and were followed an average of 7.4 years after initial injection. There were no safety concerns noted, no serious adverse events, and the multiple dosing regimen was well tolerated. Four of the six patients showed a measurable clinical improvement following MSC-NP treatment. DISCUSSION This pilot study supports preliminary first-in-human safety and tolerability of autologous MSC-NP treatment for MS.

Phase I Trial of Intrathecal Mesenchymal Stem Cell-derived Neural Progenitors in Progressive Multiple Sclerosis

Background Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system and is one of the leading causes of disability in young adults. Cell therapy is emerging as a therapeutic strategy to promote repair and regeneration in patients with disability associated with progressive MS. Methods We conducted a phase I open-label clinical trial investigating the safety and tolerability of autologous bone marrow mesenchymal stem cell-derived neural progenitor (MSC-NP) treatment in 20 patients with progressive MS. MSC-NPs were administered intrathecally (IT) in three separate doses of up to 1 × 107 cells per dose, spaced three months apart. The primary endpoint was to assess safety and tolerability of the treatment. Expanded disability status scale (EDSS), timed 25-ft walk (T25FW), muscle strength, and urodynamic testing were used to evaluate treatment response. This trial is registered with ClinicalTrials.gov, number NCT01933802. Findings IT MSC-NP treatment was safe and well tolerated. The 20 enrolled subjects completed all 60 planned treatments without serious adverse effects. Minor adverse events included transient fever and mild headaches usually resolving in <24 h. Post-treatment disability score analysis demonstrated improved median EDSS suggesting possible efficacy. Positive trends were more frequently observed in the subset of SPMS patients and in ambulatory subjects (EDSS ≤ 6.5). In addition, 70% and 50% of the subjects demonstrated improved muscle strength and bladder function, respectively, following IT MSC-NP treatment. Interpretation The possible reversal of disability that was observed in a subset of patients warrants a larger phase II placebo-controlled study to establish efficacy of IT MSC-NP treatment in patients with MS. Funding source The Damial Foundation.

Biomarkers of multiple sclerosis: current findings

Multiple sclerosis (MS) is an autoimmune disease affecting the brain and spinal cord that is associated with chronic inflammation leading to demyelination and neurodegeneration. With the recent increase in the number of available therapies for MS, optimal treatment will be based on a personalized approach determined by an individual patient’s prognosis and treatment risks. An integral part of such therapeutic decisions will be the use of molecular biomarkers to predict disability progression, monitor ongoing disease activity, and assess treatment response. This review describes current published findings within the past 3 years in biomarker research in MS, specifically highlighting recent advances in the validation of cerebrospinal fluid biomarkers such as neurofilaments (light and heavy chains), chitinases and chitinase 3-like proteins, soluble surface markers of innate immunity, and oligoclonal immunoglobulin M antibodies. Current research in circulating miRNAs as biomarkers of MS is also discussed. Continued validation and testing will be required before MS biomarkers are routinely applied in a clinical setting.

Fetuin-A deficiency protects mice from Experimental Autoimmune Encephalomyelitis (EAE) and correlates with altered innate immune response

Fetuin-A is a biomarker of disease activity in multiple sclerosis. Our aim was to investigate whether Fetuin-A plays a direct role in the neuroinflammatory response in the mouse EAE model. Peak Fetuin-A expression in the CNS and in peripheral lymphoid tissue correlated with peak EAE disease activity. Fetuin-A-deficient mice showed reduced EAE severity associated with an accumulation of splenic monocyte and dendritic cell populations, increased IL-12p40, ASC1, and IL-1β expression, and an increase in T regulatory cells. The upregulation of Fetuin-A in LPS-stimulated dendritic cells and microglia further supports an intrinsic role of Fetuin-A in regulating innate immune activation during EAE.